The present invention provides a genus of guanylhydrazone-substituted compounds that are useful for treating various diseases associated with T cell activation and retroviral infection. The present invention is based upon the mechanistic discovery that the p38 mitogen activated protein kinase (MAPK) signaling pathway is inhibited by guanylhydrazone-substituted compounds.
The human immunodeficiency virus (HIV) has been implicated as the primary cause of the slowly degenerative immune system disease termed acquired immune deficiency syndrome (AIDS) (Barre-Sinoussi et al., Science 220:868-870, 1983; Gallo et al., Science 224:500-503, 1984). In humans, HIV replication occurs prominently in CD4+T lymphocyte populations, and HIV-infection leads to depletion of this cell type and eventually to immune incompetence, opportunistic infections, neurological dysfunctions, neoplastic growth, and ultimately death. HIV is a member of the lentivirus family of retroviruses (Teich et al., RNA Tumor Viruses, 1984, Weiss et al., eds., CSH-Press, pp. 949-956). Other retroviruses include, for example, oncogenic viruses such as human T cell leukemia viruses (HTLV-I,-II,-III), and feline leukemia virus.
HIV-infection is pandemic and HIV-associated diseases represent a major world health problem. Although considerable effort is being put into the design of effective therapeutics, currently no curative anti-retroviral drugs against AIDS exist. For example, virally encoded reverse transcriptase has been one focus of drug development. A number of reverse-transcriptase-targeted drugs, including 2xe2x80x2,3xe2x80x2-dideoxynucleoside analogs such as AZT ddI, ddC, 3TC, and d4T have been developed which have been shown to be active against HIV (Mitsuya et al., Science, 249:1533-1544 (1990). While beneficial, these nucleoside analogs are not curative (Larder et al., Science, 243:1731-1734 (1989). In addition, the drugs often cause toxic side effects such as bone marrow suppression, vomiting, and liver function abnormalities.
The late stages of HIV replication, which involve crucial virus-specific processing of certain viral encoded proteins, have also been suggested as possible anti-HIV drug targets. Late stage processing is dependent on the activity of a viral protease, and drugs are marketed which inhibit this protease (Erickson, Science 249:527-533, 1990). Thus, although a great deal of effort is being directed to the design and testing of anti-retroviral drugs, effective, non-toxic treatments are still needed.
Mitogen Activated Protein (MAP) kinases are important mediators of signal transduction from the cell surface to the nucleus. The ERK1 and ERK2 mammalian subtype of the MAP kinase family have been cloned. Two additional subtypes have been discovered, p38 MAP kinase and c-jun kinase (JNK) which may be activated independently and simultaneously. The ERK pathway is activated by growth factors or phorbol esters (Marshall, Cell 80:179-185, 1995). In contrast, p38 MAP kinase and JNK pathways are activated by inflammatory cytokines and cellular stresses such as heat shock, osmotic stress or ultraviolet light (Galcheva-Gargova et al., Science 265:806-808, 1994; Kyriakis et al., Nature 369:156-160, 1994; and Raingeaud et al., J. Biol. Chem. 270:7420-7426, 1995).
Mammalian p38 MAP kinase was identified in murine pre-B cells transfected with the LPS-complex receptor, CD 14, and in murine macrophages where it is activated in response to LPS (Han et al., Science 265:808-811, 1994). p38 has been identified as the mammalian homologue of yeast osmosensing MAP kinase, HOG1 (Brewster et al., Science 259:1760-1763, 1993), and the Xenopus kinase Mpk2 (Rouse et al., Cell 78:1027-1037, 1994). CSBP1 and CSBP2 have been identified as human homologues of murine p38 MAP kinase (Lee et al., Nature 372:739-746, 1994). p38 MAPK activation has also been identified in lymphocytes during intra-thymic signaling essential for the differentiation and repertoire selection of mature T-cell development (Sen et al., J. Immunol. 156:4535, 1996). Once activated by phosphorylation, p38 MAPK acts both transcriptionally and translationally to phosphorylate downstream targets. Such targets include the transcription factors ATF-2, CHOP, HSP27, Max (Raingeaud et al., J. Biol. Chem. 270:7420, 1995; Batchvarova et al., EMBO J. 14:4654, 1995; Freshney et al., Cell 78:1039, 1994; and Zervos et al., Proc. Natl. Acad. Sci. USA 92:10531, 1995), and the protein kinases MAPK activated protein kinase-2 and -3 (MAPKAP-K2 and-K3) (McLaughlin et al., J. Biol. Chem. 271: 8488, 1996; and Beyaert et al., EMBO J. 15:1914, 1996).
p38 MAP kinase is activated in TNF-treated cells and plays a selective role in gene induction, controlling, for instance, synthesis of IL-6 and granulocyte macrophage colony stimulating factor (GM-CSF) (Beyart et al., EMBO J. 15:1914-1923, 1996). The role of p38 in the signaling pathway for cytokine responses was further shown in studies using model systems of cytokine induction of HIV gene expression constructs to measure the effects of inhibiting p38. Using a laboratory cell line chronically infected with latent HIV, certain pharmaceutical inhibitors of p38 MAPK were reported to block the cytokine-induced production of HIV p24 (Shapiro et al., Eur. Cytokine Netw. 7:557, 1996). p38 inhibitors were also reported to block the cytokine-specific induction of HIV LTR-driven expression of an unrelated reporter molecule in a transfection model system (Kumar et al., Eur. Cytokine Netw. 7:558, 1996).
In view of the multitude of roles played in intracellular signaling by p38 MAP kinase, there is a need in the art to find compounds that will inhibit intracellular signaling pathways through p38 MAP kinase. Such selective inhibitors of this signaling pathway will inhibit T cell activation and therefore show therapeutic utility for treating infections caused by retroviruses (for example, HIV) and for various autoimmune diseases (for example, rheumatoid arthritis, lupus, graft versus host, host versus graft, insulin-dependent diabetes, and multiple sclerosis).
The present invention provides therapeutic modalities for the treatment of diseases and disorders caused as a result of T cell activation, in particular HIV-infection, by virtue of the p38 MAPK cellular signaling pathway as a target of intervention. The present invention specifically provides guanylhydrazone-substituted compounds to treat disorders and diseases related to T cell activation and HIV-infection. The present invention also relates to the use of other compounds which inhibit the p38 MAP kinase pathway in treating immune diseases, and other diseases or clinical conditions in which T lymphocytes and cytokines and/or other mediators released by activated T lymphocytes are implicated in the pathology of the disease condition.
The present invention also relates to therapeutic modalities for the treatment of disorders and diseases related to T cell activation, in particular HIV-infection, by administering an effective amount of a p38 MAPK pathway inhibitor in combination with at least one other therapeutic agent. Preferably the p38 MAPK pathway inhibitor is used in combination therapy with an antiviral agent of another class (i.e., treatment mechanism of action), such as a reverse transcriptase inhibitor (e.g., AZT, ddI, ddC, 3TC); an HIV protease inhibitor (e.g., ABT-538); or a preintegration complex inhibitor.
The invention is based on the discovery that the p38 MAPK signal transduction pathway participates in T lymphocyte co-stimulation. p38 MAPK activity was found to be enhanced in human T lymphocytes stimulated with a-CD3 and a CD28. Inhibition of the p38 MAPK pathway, by the addition of an exemplary multivalent guanylhydrazone, CNI-1493, resulted in inhibition of activation of the T lymphocytes, as measured by 1L-2 synthesis.
The various modalities of treatment described herein were designed on the basis of the proposed model. In particular, multivalent guanylhydrazone compounds, small organic molecules, antisense molecules, ribozymes and triple helix molecules targeted to the p38 MAP kinase pathway may be used to inhibit T cell activation and HIV-infection. In a specific embodiment of the invention, multivalent guanylhydrazone compounds, such as CNI-1493, may be used to inhibit HIV-infection. In yet another embodiment of the invention, upstream and downstream components of the p38 MAP kinase pathway may be targeted to inhibit HIV-infection. For example, MAPKAP kinase-2, which is downstream of p38 MAP kinase, may be targeted by antisense, ribozyme or triple helix molecules, to inhibit HIV-infection.
The present invention further relates to screening assays to identify compounds which inhibit the p38 MAP kinase pathway and may be used to treat diseases or disorders related to T cell activation, in particular HIV-infection.